Approximately 50% of human tumors express wild type p53 that is functionally compromised due to defects in regulation. P53 level and activity is regulated by MDM2 and MDMX. MDM2 is a validated drug target and novel inhibitors are being developed, including the recently reported MDM2/p53 binding disrupter Nutlin 3. MDMX is frequently overexpressed in tumors, but its significance in p53 inactivation is relatively unknown. We found that MDMX is an important regulator of p53 response to abnormal ribosomal biogenesis. P53 activation by ribosomal stress agents such as Actinomycin D (Act.D) and 5-fluorouracil (5-FU) requires down-regulation of MDMX by accelerated degradation. Tumor cells overexpressing MDMX are less sensitive to Act.D and 5-FU-induced growth arrest, whereas knockdown of MDMX increases sensitivity to ribosomal stress and abrogates tumor formation in nude mice. Surprisingly, Nutlin 3 does not disrupt p53-MDMX binding and functions inefficiently in MDMX overexpressing cells, suggesting a need to develop MDMX- specific or MDM2 and MDMX dual inhibitors. We hypothesize that both MDM2 and MDMX are important targets in cancer treatment due to their shared and distinct functional characteristics. Development of novel MDM2 and MDMX inhibitors are necessary for validating the therapeutic potential of targeting both MDM2 and MDMX-P53 interactions. We propose the following aims to test these hypotheses. (1)Investigate the role and mechanism of MDMX in tumor formation. (2) Structure-based rational design of inhibitors of MDM2 and MDMX-p53 interaction. (3) Synthesis of focused chemical libraries based on lead compounds. (4) Evaluate compounds for p53 activation and anti-tumor activity. These experiments should validate MDMX as a drug target and may provide lead compounds for further development of experimental drugs against MDM2 and MDMX.